CN107727867A - A kind of preparation method of Complement C_3 detection kit - Google Patents
A kind of preparation method of Complement C_3 detection kit Download PDFInfo
- Publication number
- CN107727867A CN107727867A CN201710915596.1A CN201710915596A CN107727867A CN 107727867 A CN107727867 A CN 107727867A CN 201710915596 A CN201710915596 A CN 201710915596A CN 107727867 A CN107727867 A CN 107727867A
- Authority
- CN
- China
- Prior art keywords
- buffer solutions
- antibody
- human
- reagent
- complement
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4716—Complement proteins, e.g. anaphylatoxin, C3a, C5a
Abstract
The invention discloses a kind of preparation method of Complement C_3 detection kit, comprise the following steps：(1) reagent R1 constituent content is pressed, is configured to R1 buffer solutions；PEG 8000, EDTA Na, Sodium azide are dissolved in R1 buffer solutions, then pH to 8.5 is adjusted with 1mol/L hydrochloric acid or sodium hydroxide, obtains reagent R1；(2) reagent R2 constituent content is pressed, prepares R2 buffer solutions；BSA, EDTA Na, Tween 20, network albumen, sucrose, NaCl, Sodium azide are dissolved in R2 buffer solutions, obtain R2 dispersion liquids；Prepare the coated anti-human C3 antibody of polystyrene latex particulate；PH to 7.4 is adjusted with the coated anti-human C3 antibody of R2 dispersion liquid dissolved polystyrene emulsion particles, then with 1mol/L hydrochloric acid or sodium hydroxide, obtains reagent R2.Advantage of the present invention is：The kit being prepared has higher sensitivity and stability, and simple to operate, quick.
Description
Technical field
The present invention relates to technical field of medical examination, more particularly to a kind of preparation method of Complement C_3 detection kit.
Background technology
Complement C_3 is made up of two peptide chains of α and β Disulfide bonds link, is the globulin of β 1, molecular weight 180kD, containing sugar
Amount accounts for 2.2%, is the complement component that content is most in serum, accounts for more than the 1/3 of total complement content.In complement system activity
During, either classical pathway or alternative pathway, it is both needed to promote after C3 activation follow-up complement component (C5-C9) company
Lock reactor.Therefore, it plays a crucial role in two activated pathway.Complement C_3 is mainly synthesized secretion in hepatic parenchymal cells, few
Amount is synthesized by macrophage and monocyte.Under physiological conditions, proteolytic enzyme in body fluid or existing for tissue inflammation position can
Slowly cracking C3, persistently produces a small amount of C3b and C3 convertase (C3bB), typically can be by FI, H factor rapid inactivations, therefore simultaneously
Not activating complement system, the substance activating once C3 is activated, C3b can synthesize new C3bBb simultaneously under Factor B, the effect of the D factors again
C3 further is activated, cracks, discharge many biological active fragments, its result can behave as strengthening the defence capability of body,
Also it may occur in which that the immunopathogenesis for causing disease acts on.
Increasing for C3 is substantially similar to complement activity but more sensitive to reduction.Complement C_3 body tissue damage and
During acute inflammation, often increase or be normal, such as bacteremia, pneumonia, tonsillitis, tuberculosis, typhoid fever, measles, epidemic meningitis；Tumour is suffered from
Person, especially with liver cancer, the rise of the content of change of serum C 3 is more notable, but cancer of pancreas late period then becomes with recessive lymphocytic leukemia in reduction
Gesture.Complement dynamic change is clinically increasingly taken seriously.Gastritis total complement of serum caused by antigen-antibody complex
It is decreased obviously with C3.The reduction of most of SLE Patients serum complements and that sb.'s illness took a turn for the worse is relevant.Activity is complete
C1, C4, C2 and C3 are reduced in body patients with SLE serum, and serum complement is horizontal during disease amelioration recovers normal.Infectious disease
And when tissue damage and acute inflammation, C2, C3, C4 increase, complement activity is normal or raises, but late period then reduces.Tumour
Patient's complement amount raises, particularly liver cancer, and C3 rises are the most obvious, have diagnostic significance.Cancer of pancreas late period and recessive lymph are thin
Born of the same parents leukaemic C3 is reduced.Patients with liver diseases C3 is mostly to reduce, renal transplant patient C3 horizontal instability, and C3 is anti-in rejection
Raise, be then lowered into normal following when should start.
At present, the content method for detecting serum complement C3 is mainly common immunoturbidimetry, and content of complement C 3 is detected
Kit it is also mostly be to be designed based on the method.Immunoturbidimetry is antigen-antibody combination dynamic assay method, its general principle
It is：When antigen and antibody react in certain dilution system and ratio is suitable, the soluble immune complex of formation is diluting
In the presence of the poly- agent of rush in system, separated out from liquid phase, form particulate, reaction solution turbidity is occurred；When antibody concentration is fixed,
The amount of the immune complex of formation increases with the increase of amount of antigen in sample, and the turbidity of reaction solution is consequently increased；Pass through
The turbidity of measure reaction solution compares with series of standards product, you can calculates the content of antigen in sample.However, this method but also
In the presence of some drawbacks：Sensitivity is low, the range of linearity is narrow, poor repeatability and unstable.
Latex particle enhancing turbidimetry (PETIA) is that occur in recent years a kind of is relatively stable, accurate body fluid albumen is homogeneous
Immunoturbidimetry.PETIA methods are broadly divided into two kinds：One kind is scattered light urbidmetry；One kind is turbidimetry；Both approaches
General principle is similar, is all the surface-crosslinked corresponding antibodies using polymer latex microballoon as carrier, when the microballoon for being crosslinked with antibody
It after antigen binding, can rapidly assemble in the short time, change the astigmatism performance or light transmission of reaction solution, and reaction solution astigmatism
The change of energy or light transmission is related to the concentration of tested antigen, can reflect the amount of tested antigen within the specific limits.
The latex particle that PETIA methods use is mostly inert microspheres, carboxyl microballoon and amino microballoon；The carboxyl of microsphere surface or
The aminoterminal covalent bond of amino and antibody, the bridging chemistry arm of formation reduce steric effect, not only increase the knot of antibody
Conjunction rate, and suitable three dimensions stereochemical structure is provided for antibody, effectively protect the active region of antibody and antigen binding；It is micro-
Ball particle diameter typically in 20nm-4um, improves detection sensitivity.The release of microballoon can solve clinic to a certain extent
The problems such as range of linearity of detection is narrow, disturbing factor is more, stable experiment difference.
Therefore, it is badly in need of a kind of preparation method of the Complement C_3 detection kit based on latex particle enhancing turbidimetry at present.
The content of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of detection speed is fast, high sensitivity, linear
The preparation method of wide, reproducible and stabilization the Complement C_3 detection kit of scope.
The present invention is achieved by the following technical solutions：A kind of preparation method of Complement C_3 detection kit, including such as
Lower step：
(1) according to following component content reagent preparation R1：
Reagent R1：
Its solvent is purified water；
1. according to mentioned reagent R1 constituent content, Tris is dissolved in purified water, stirred, treats fully to dissolve, matches somebody with somebody
R1 buffer solutions are made；
2. according to mentioned reagent R1 constituent content, PEG 8000, EDTA-Na, Sodium azide are dissolved in R1 buffer solutions
In, stir, then adjust pH to 8.5 with 1mol/L hydrochloric acid or sodium hydroxide, that is, reagent R1 is made；
(2) according to following component content reagent preparation R2：
Reagent R2：
Its solvent is purified water；
1. according to mentioned reagent R2 constituent content, Tris is dissolved in purified water, stirred, treats fully to dissolve, matches somebody with somebody
R2 buffer solutions are made；
2. according to mentioned reagent R2 constituent content, by BSA, EDTA-Na, Tween-20, network albumen, sucrose, NaCl, folded
Nitrogen sodium is dissolved in R2 buffer solutions, is stirred, and obtains R2 dispersion liquids；
3. prepare the coated anti-human C3 antibody of polystyrene latex particulate：
A. it is 1%-5% polystyrene latex to be diluted into concentration with PB buffer solutions, is added in every milliliter of bulk solution
EDAC 1.6mg, 2h is reacted at room temperature, 30-40min is centrifuged under 10000-20000rpm rotating speeds, removes supernatant, precipitation is suspended in MES
In buffer solution, ultrasonic disperse；
B. 30-40min is centrifuged under 10000-20000rpm rotating speeds, abandons supernatant, precipitation is suspended in PB buffer solutions,
It is 3.0% to make polystyrene latex particulate ultimate density therein, ultrasonic disperse；Then, isometric contain is added while stirring
The PB dilutions of anti-human C3 antibody, mix, react at room temperature 2h, latex particle ultimate density therein is 1.5%；
C.10000-20000rpm 30-40min is centrifuged under rotating speed, abandons supernatant, precipitated in suspension PB buffer solutions, ultrasound point
Dissipate, add BSA, 4 DEG C of closings overnight, after supernatant is removed in centrifugation, obtain the coated anti-human C3 of finally required polystyrene latex particulate
Antibody；
4. dissolving the obtained coated anti-human C3 antibody of polystyrene latex particulate with R2 dispersion liquids, make latex therein
The final concentration of 0.1-2.0% of particle, ultrasonic disperse, then pH to 7.4 is adjusted with 1mol/L hydrochloric acid or sodium hydroxide, reagent is finally made
R2。
One of preferred embodiment as the present invention, the step for preparing the coated anti-human C3 antibody of polystyrene latex particulate
In rapid a, polystyrene latex particulate that the polystyrene latex particulate starting material that uses is 100-200nm for particle diameter.
One of preferred embodiment as the present invention, the step for preparing the coated anti-human C3 antibody of polystyrene latex particulate
In rapid a, the PB buffer solutions used is 0.1-0.15mol/L PB buffer solutions, and the MES buffer solutions used is 0.05-0.1mmol/L
MES buffer solutions.
One of preferred embodiment as the present invention, the step for preparing the coated anti-human C3 antibody of polystyrene latex particulate
In rapid b, the PB buffer solutions used is 0.1-0.15mol/L PB buffer solutions, and the PB dilutions used is 0.02-0.05mol/L
PB buffer solutions.
One of preferred embodiment as the present invention, the step for preparing the coated anti-human C3 antibody of polystyrene latex particulate
In rapid b, anti-human C3 antibody and the coated mass ratio of polystyrene latex particulate are (1-3)：10.
One of preferred embodiment as the present invention, the step for preparing the coated anti-human C3 antibody of polystyrene latex particulate
In rapid c, the PB buffer solutions that use for 0.02-0.05mol/L PB buffer solutions.
One of preferred embodiment as the present invention, the step for preparing the coated anti-human C3 antibody of polystyrene latex particulate
In rapid c, the BSA used is 0.5%-1%BSA.
The present invention compared with prior art the advantages of be：
(1) kit for preparing of the present invention is simple to operate, quick, from detecting out that result only needs 10 minutes；
(2) Immunoturbidimetry quantitative determination Complement C_3 has good amplification, and measure solution turbidity increases
Content of complement C 3 in the degree and sample that add is proportionate, and substantially increases the sensitivity of reagent detection, the linear model of kit
Width is enclosed, it is reproducible；Kit prepared by the present invention experiments verify that, sensitivity reaches 3.0mg/dl, and maximum concentration reaches
HOOK effects are had not yet to see during 1000.0mg/dl, repeatability≤2.3% in batch, repeatability≤6.7% between batch；
(3) kit for preparing of the present invention can be stablized at 2-8 DEG C preserves 24 months, 37 DEG C of accelerated tests 7 days, as a result shows
Degradation rate is 6.3%；
(4) kit prepared by the present invention can be applied on automatic clinical chemistry analyzer, is adapted to full-automatic testing, can advise greatly
The development and popularization of mould.
Embodiment
Embodiments of the invention are elaborated below, the present embodiment is carried out lower premised on technical solution of the present invention
Implement, give detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following implementation
Example.
Embodiment 1
A kind of preparation method of Complement C_3 detection kit of the present embodiment, comprises the following steps：
(1) according to following component content reagent preparation R1：
Reagent R1：
Its solvent is purified water；
1. according to mentioned reagent R1 constituent content, Tris is dissolved in purified water, stirred, treats fully to dissolve, matches somebody with somebody
R1 buffer solutions are made；
2. according to mentioned reagent R1 constituent content, PEG 8000, EDTA-Na, Sodium azide are dissolved in R1 buffer solutions
In, stir, then adjust pH to 8.5 with 1mol/L hydrochloric acid or sodium hydroxide, that is, reagent R1 is made；
(2) according to following component content reagent preparation R2：
Its solvent is purified water；
1. according to mentioned reagent R2 constituent content, Tris is dissolved in purified water, stirred, treats fully to dissolve, matches somebody with somebody
R2 buffer solutions are made；
2. according to mentioned reagent R2 constituent content, by BSA, EDTA-Na, Tween-20, network albumen, sucrose, NaCl, folded
Nitrogen sodium is dissolved in R2 buffer solutions, is stirred, and obtains R2 dispersion liquids；
3. prepare the coated anti-human C3 antibody of polystyrene latex particulate：
A. it is 1% the polystyrene latex that particle diameter is 100nm to be diluted into concentration with 0.1mol/L PB buffer solutions, per milli
Rise and EDAC 1.6mg are added in bulk solution, react at room temperature 2h, centrifuge 30min under 10000rpm rotating speeds, remove supernatant, precipitation is outstanding
Float in 0.05mmol/L MES buffer solutions, ultrasonic disperse；
B. 30min is centrifuged under 10000rpm rotating speeds, abandons supernatant, precipitation is suspended in 0.1mol/L PB buffer solutions
In, it is 3.0% to make polystyrene latex particulate ultimate density therein, ultrasonic disperse；Then, add while stirring in equal volume
0.02mol/L PB dilutions containing anti-human C3 antibody, mix, react at room temperature 2h, latex particle ultimate density therein is
1.5%；Wherein, anti-human C3 antibody and the coated mass ratio of polystyrene latex particulate are 1：10；
C.10000-20000rpm 30min is centrifuged under rotating speed, abandons supernatant, precipitation is suspended in 0.02mol/L PB buffer solutions
In, ultrasonic disperse adds 0.5%BSA, and 4 DEG C of closings overnight, after supernatant is removed in centrifugation, it is micro- to obtain finally required polystyrene latex
The coated anti-human C3 antibody of grain；
4. dissolving the obtained coated anti-human C3 antibody of polystyrene latex particulate with R2 dispersion liquids, make latex therein
Particle final concentration of 0.1%, ultrasonic disperse, then pH to 7.4 is adjusted with 1mol/L hydrochloric acid or sodium hydroxide, reagent R2 is finally made.
Embodiment 2
A kind of preparation method of Complement C_3 detection kit of the present embodiment, comprises the following steps：
(1) according to following component content reagent preparation R1：
Reagent R1：
Its solvent is purified water；
1. according to mentioned reagent R1 constituent content, Tris is dissolved in purified water, stirred, treats fully to dissolve, matches somebody with somebody
R1 buffer solutions are made；
2. according to mentioned reagent R1 constituent content, PEG 8000, EDTA-Na, Sodium azide are dissolved in R1 buffer solutions
In, stir, then adjust pH to 8.5 with 1mol/L hydrochloric acid or sodium hydroxide, that is, reagent R1 is made；
(2) according to following component content reagent preparation R2：
Reagent R2：
Its solvent is purified water；
1. according to mentioned reagent R2 constituent content, Tris is dissolved in purified water, stirred, treats fully to dissolve, matches somebody with somebody
R2 buffer solutions are made；
2. according to mentioned reagent R2 constituent content, by BSA, EDTA-Na, Tween-20, network albumen, sucrose, NaCl, folded
Nitrogen sodium is dissolved in R2 buffer solutions, is stirred, and obtains R2 dispersion liquids；
3. prepare the coated anti-human C3 antibody of polystyrene latex particulate：
A. it is 5% the polystyrene latex that particle diameter is 200nm to be diluted into concentration with 0.15mol/L PB buffer solutions, often
EDAC 1.6mg are added in milliliter bulk solution, 2h is reacted at room temperature, centrifuges 40min under 20000rpm rotating speeds, remove supernatant, will precipitate
It is suspended in 0.1mmol/L MES buffer solutions, ultrasonic disperse；
B. 40min is centrifuged under 20000rpm rotating speeds, abandons supernatant, precipitation is suspended in 0.15mol/L PB buffer solutions
In, it is 3.0% to make polystyrene latex particulate ultimate density therein, ultrasonic disperse；Then, add while stirring in equal volume
0.05mol/L PB dilutions containing anti-human C3 antibody, mix, react at room temperature 2h, latex particle ultimate density therein is
1.5%；Wherein, anti-human C3 antibody and the coated mass ratio of polystyrene latex particulate are 3：10；
C.20000rpm 40min is centrifuged under rotating speed, abandons supernatant, precipitation is suspended in 0.05mol/L PB buffer solutions, ultrasound
It is scattered, 1%BSA is added, 4 DEG C of closings overnight, after supernatant is removed in centrifugation, it is coated anti-to obtain finally required polystyrene latex particulate
People's C3 antibody；
4. dissolving the obtained coated anti-human C3 antibody of polystyrene latex particulate with R2 dispersion liquids, make latex therein
Particle final concentration of 2.0%, ultrasonic disperse, then pH to 7.4 is adjusted with 1mol/L hydrochloric acid or sodium hydroxide, reagent R2 is finally made.
Embodiment 3
A kind of preparation method of Complement C_3 detection kit of the present embodiment, comprises the following steps：
(1) according to following component content reagent preparation R1：
Reagent R1：
Its solvent is purified water；
1. according to mentioned reagent R1 constituent content, Tris is dissolved in purified water, stirred, treats fully to dissolve, matches somebody with somebody
R1 buffer solutions are made；
2. according to mentioned reagent R1 constituent content, PEG 8000, EDTA-Na, Sodium azide are dissolved in R1 buffer solutions
In, stir, then adjust pH to 8.5 with 1mol/L hydrochloric acid or sodium hydroxide, that is, reagent R1 is made；
(2) according to following component content reagent preparation R2：
Reagent R2：
Its solvent is purified water；
1. according to mentioned reagent R2 constituent content, Tris is dissolved in purified water, stirred, treats fully to dissolve, matches somebody with somebody
R2 buffer solutions are made；
2. according to mentioned reagent R2 constituent content, by BSA, EDTA-Na, Tween-20, network albumen, sucrose, NaCl, folded
Nitrogen sodium is dissolved in R2 buffer solutions, is stirred, and obtains R2 dispersion liquids；
3. prepare the coated anti-human C3 antibody of polystyrene latex particulate：
A. it is 3% the polystyrene latex that particle diameter is 150nm to be diluted into concentration with 0.13mol/L PB buffer solutions, often
EDAC 1.6mg are added in milliliter bulk solution, 2h is reacted at room temperature, centrifuges 35min under 15000rpm rotating speeds, remove supernatant, will precipitate
It is suspended in 0.08mmol/L MES buffer solutions, ultrasonic disperse；
B. 35min is centrifuged under 15000rpm rotating speeds, abandons supernatant, precipitation is suspended in 0.13mol/L PB buffer solutions
In, it is 3.0% to make polystyrene latex particulate ultimate density therein, ultrasonic disperse；Then, add while stirring in equal volume
0.03mol/L PB dilutions containing anti-human C3 antibody, mix, react at room temperature 2h, latex particle ultimate density therein is
1.5%；Wherein, anti-human C3 antibody and the coated mass ratio of polystyrene latex particulate are 2：10；
C.15000rpm 35min is centrifuged under rotating speed, abandons supernatant, precipitation is suspended in 0.04mol/L PB buffer solutions, ultrasound
It is scattered, 0.8%BSA is added, 4 DEG C of closings overnight, after supernatant is removed in centrifugation, it is coated to obtain finally required polystyrene latex particulate
Anti-human C3 antibody；
4. dissolving the obtained coated anti-human C3 antibody of polystyrene latex particulate with R2 dispersion liquids, make latex therein
Particle final concentration of 1%, ultrasonic disperse, then pH to 7.4 is adjusted with 1mol/L hydrochloric acid or sodium hydroxide, reagent R2 is finally made.
Embodiment 4
The application method of the Complement C_3 detection kit being prepared using above-described embodiment method of the present embodiment, including
Following steps：
(1) 10uL testing samples are mixed with 150uL reagents R1,37 DEG C of incubation 5min；
(2) mixed with 50uL reagents R2,37 DEG C of reaction 5min；
(3) reacted absorbance A is determined at 600nm wavelength with automatic clinical chemistry analyzer；
(4) concentration of the Complement C_3 in sample is calculated according to the absorbance A of reading.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should be included in the scope of the protection.
Claims (7)
1. a kind of preparation method of Complement C_3 detection kit, it is characterised in that comprise the following steps：
(1) according to following component content reagent preparation R1：
Reagent R1：
Its solvent is purified water；
1. according to mentioned reagent R1 constituent content, Tris is dissolved in purified water, stirred, treats fully to dissolve, is configured to
R1 buffer solutions；
2. according to mentioned reagent R1 constituent content, PEG 8000, EDTA-Na, Sodium azide are dissolved in R1 buffer solutions, stirred
Mix uniformly, then pH to 8.5 is adjusted with 1mol/L hydrochloric acid or sodium hydroxide, that is, reagent R1 is made；
(2) according to following component content reagent preparation R2：
Reagent R2：
Its solvent is purified water；
1. according to mentioned reagent R2 constituent content, Tris is dissolved in purified water, stirred, treats fully to dissolve, is configured to
R2 buffer solutions；
2. according to mentioned reagent R2 constituent content, by BSA, EDTA-Na, Tween-20, network albumen, sucrose, NaCl, Sodium azide
It is dissolved in R2 buffer solutions, stirs, obtains R2 dispersion liquids；
3. prepare the coated anti-human C3 antibody of polystyrene latex particulate：
A. it is 1%-5% polystyrene latex to be diluted into concentration with PB buffer solutions, and EDAC is added in every milliliter of bulk solution
1.6mg, 2h is reacted at room temperature, 30-40min is centrifuged under 10000-20000rpm rotating speeds, removes supernatant, precipitation is suspended in MES bufferings
In liquid, ultrasonic disperse；
B. 30-40min is centrifuged under 10000-20000rpm rotating speeds, abandons supernatant, precipitation is suspended in PB buffer solutions, makes it
In polystyrene latex particulate ultimate density be 3.0%, ultrasonic disperse；Then, what addition while stirring was isometric contains anti-human
The PB dilutions of C3 antibody, mix, react at room temperature 2h, latex particle ultimate density therein is 1.5%；
C.10000-20000rpm 30-40min is centrifuged under rotating speed, abandons supernatant, precipitated in suspension PB buffer solutions, ultrasonic disperse, add
Enter BSA, 4 DEG C of closings overnight, after supernatant is removed in centrifugation, obtain the coated anti-human C3 antibody of finally required polystyrene latex particulate；
4. dissolving the obtained coated anti-human C3 antibody of polystyrene latex particulate with R2 dispersion liquids, make latex particle therein
Final concentration of 0.1-2.0%, ultrasonic disperse, then pH to 7.4 is adjusted with 1mol/L hydrochloric acid or sodium hydroxide, reagent R2 is finally made.
2. the preparation method of Complement C_3 detection kit according to claim 1, it is characterised in that described to prepare polyphenyl second
In the step a of the coated anti-human C3 antibody of alkene emulsion particle, the polystyrene latex particulate starting material that uses is 100- for particle diameter
200nm polystyrene latex particulate.
3. the preparation method of Complement C_3 detection kit according to claim 1, it is characterised in that described to prepare polyphenyl second
In the step a of the coated anti-human C3 antibody of alkene emulsion particle, the PB buffer solutions that use for 0.1-0.15mol/L PB buffer solutions,
The MES buffer solutions used for 0.05-0.1mmol/L MES buffer solutions.
4. the preparation method of Complement C_3 detection kit according to claim 1, it is characterised in that described to prepare polyphenyl second
In the step b of the coated anti-human C3 antibody of alkene emulsion particle, the PB buffer solutions that use for 0.1-0.15mol/L PB buffer solutions,
The PB dilutions used for 0.02-0.05mol/L PB buffer solutions.
5. the preparation method of Complement C_3 detection kit according to claim 1, it is characterised in that described to prepare polyphenyl second
In the step b of the coated anti-human C3 antibody of alkene emulsion particle, anti-human C3 antibody and the coated mass ratio of polystyrene latex particulate
For (1-3)：10.
6. the preparation method of Complement C_3 detection kit according to claim 1, it is characterised in that described to prepare polyphenyl second
In the step c of the coated anti-human C3 antibody of alkene emulsion particle, the PB buffer solutions that use for 0.02-0.05mol/L PB buffer solutions.
7. the preparation method of Complement C_3 detection kit according to claim 1, it is characterised in that described to prepare polyphenyl second
In the step c of the coated anti-human C3 antibody of alkene emulsion particle, the BSA used is 0.5%-1%BSA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710915596.1A CN107727867A (en) | 2017-09-30 | 2017-09-30 | A kind of preparation method of Complement C_3 detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710915596.1A CN107727867A (en) | 2017-09-30 | 2017-09-30 | A kind of preparation method of Complement C_3 detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107727867A true CN107727867A (en) | 2018-02-23 |
Family
ID=61208397
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710915596.1A Withdrawn CN107727867A (en) | 2017-09-30 | 2017-09-30 | A kind of preparation method of Complement C_3 detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107727867A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111693709A (en) * | 2019-03-12 | 2020-09-22 | 程明 | Complement C1q determination kit and determination method thereof |
| CN114047338A (en) * | 2021-11-10 | 2022-02-15 | 上海捷门生物技术有限公司 | Urine transferrin detection kit and detection method thereof |
-
2017
- 2017-09-30 CN CN201710915596.1A patent/CN107727867A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111693709A (en) * | 2019-03-12 | 2020-09-22 | 程明 | Complement C1q determination kit and determination method thereof |
| CN114047338A (en) * | 2021-11-10 | 2022-02-15 | 上海捷门生物技术有限公司 | Urine transferrin detection kit and detection method thereof |
| CN114047338B (en) * | 2021-11-10 | 2024-03-12 | 上海捷门生物技术有限公司 | Urine transferrin detection kit and detection method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5788330B2 (en) | Organic colored fine particles, diagnostic kit containing the same, and in vitro diagnostic method | |
| JP5351054B2 (en) | Method for producing insoluble carrier particles, insoluble carrier particles, measurement reagent, sample analysis tool, and immunoturbidimetric method | |
| EP0064274B1 (en) | Method for assaying antigen-antibody reactions and reagent therefor | |
| CN102662064A (en) | Immunonephelometry kit for detecting lipid carrier protein related to neutrophils gelatinase and preparation method thereof | |
| CN111856016A (en) | Saliva liquefaction sugar chain antigen determination kit and preparation method thereof | |
| CN107727867A (en) | A kind of preparation method of Complement C_3 detection kit | |
| CN111320696A (en) | MMP-3 antibody compound based on streptavidin latex and kit thereof | |
| JP4086266B2 (en) | Immunoassay method | |
| CN104215778B (en) | Crosslinked magnetic particle of a kind of C peptide monoclonal antibody and preparation method thereof and the detection kit that comprises it | |
| CN109490533B (en) | Pepsinogen II (PG II) detection kit and production process thereof | |
| CN107741501A (en) | A kind of Complement C_3 detection kit and its application method | |
| CN112730848B (en) | Kit and method for detecting retinol binding protein | |
| CN111830260B (en) | Process for reducing inter-batch difference in production of latex-enhanced immunoturbidimetry reagent | |
| JP6737643B2 (en) | Labeled antibody, method for producing the same, and immunological assay | |
| JP3471198B2 (en) | Reagent for turbidimetric determination of immunological latex | |
| JP2004325414A (en) | Method and kit for measuring immunity | |
| JPH1123573A (en) | Immunological measuring method | |
| JPH11344494A (en) | Immunological agglutination reaction reagent and method for suppressing prozone phenomenon by using the same | |
| JP2020125909A (en) | Colored cellulose fine particles with uniform particle size | |
| JP2000046828A (en) | Immunoassay reagent and production thereof | |
| JP3328053B2 (en) | Determination of antibody or antigen concentration by immunoagglutination | |
| JP7096482B2 (en) | Means for Preventing Deterioration of Immunoassay Reagents Containing Sensitized Insoluble Carrier Particles | |
| WO2020095759A1 (en) | Method for producing latex particles for measuring anti-streptolysin o antibody | |
| CN117647644A (en) | Blocking agent and application thereof in immunodetection | |
| CN115032382A (en) | Ferritin detection kit and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20180223 |
|
| WW01 | Invention patent application withdrawn after publication |